Keratin-associated Protein Genes Research Articles

A cluster of 21 keratin-associated protein genes within introns of another gene on human chromosome 21q22.3

Recently, we identified multiple unique sequences in the 21q22.3 region and predicted them to be a cluster of genes encoding hair-specific keratin-associated proteins (KAPs). Detailed computer-aided analysis of these clustered genes revealed that the cluster spans over 165 kb and consists of 21 KAP-related sequences including 16 putative genes and 5 pseudogenes. These were further divided into two subfamilies, KRTAP12 ( KRTAP12.1– 12.4 and KRTAP12.5P) and KRTAP18 ( KRTAP18.1– 18.12 and KRTAP18.13P– 18.16P). All 16 putative genes possess several intragenic repeat sequences and apparently belong to the high-sulfur KAP gene family (16–30% cysteine content) known for nonhuman mammalian species. Transcripts were detected by RT-PCR analysis for all 16 putative KAP genes and their expression was restricted to hair root cells (radix pili cells) and not found in 28 other tissues, including skin. All 16 KAP genes produced unspliced transcripts, indicating their nature to be that of active intronless genes. Interestingly, all these KAP-related genes are located within introns of the recently identified gene TSPEAR (approved gene symbol C21orf29), 214 kb in size. Surprisingly, the transcriptional direction of 8 of the 16 active genes is the same as that of C21orf29/TSPEAR. This finding suggests a novel transcription mechanism in which C21orf29/TSPEAR gene transcription passes over the multiple transcriptional termination sites of the KAP genes.

Hair Keratin Associated Proteins: Characterization of a Second High Sulfur KAP Gene Domain on Human Chromosome 21

Analysis of the EBI/GeneBank database using nonhuman hair keratin associated protein (KAP) gene sequences as a query resulted in the identification of two human KAP gene domains on chromosome 21, one of which, located at 21q22.1, has recently been characterized. The second domain presented here, an approximately 90 kb domain on chromosome 21q23, harbored 16 KAP genes and two KAP pseudogenes. By comparison with known sheep and mouse KAP families, these genes could be assigned to two KAP families, KAP10 and KAP12, with the KAP10 family (12 members) being distinctly larger than the KAP12 family (four members). Systematic cDNA/3' rapid amplification of cDNA ends isolation studies using human scalp mRNA led to the identification of eight KAP10 and two KAP12 cDNA sequences. In situ hybridization analyses of human anagen hair follicles using specific 3'-noncoding sequences of the various KAP10/KAP12 genes revealed mRNA expression of nearly all KAP10 and KAP12 members exclusively in a narrow region of the middle portion of the hair fiber cuticle. Bioinformatic analyses of the promoter regions of the KAP10/KAP12 genes demonstrated several enhancer elements that were present in nearly all of the KAP genes. Primary among these were binding elements for the ETS, heat shock factor, AML, and HOX families of transcription factors.

Characterization of human keratin-associated protein 1 family members.

Keratin-associated proteins are involved in the formation of the cross-linked network of the keratin-intermediate filament proteins that support hair fibers. In recent years, several keratin-associated protein genes have been identified and become an attractive topic in hair research. More recently, we isolated two cDNA encoding novel members of the human keratin-associated protein 1 family (human keratin-associated protein 1.6 and human keratin-associated protein 1.7), and described their expression in the hair follicle by RNA in situ hybridization. A comparison of human keratin-associated protein 1.6 and human keratin-associated protein 1.7 with other human keratin-associated protein 1 members revealed that keratin-associated protein 1 proteins are fundamentally composed of five distinct domains, and that they can be classified primarily by a striking variation in double cysteine-containing pentapeptide repeats in the repetitive I domain. The sum of the data analyzed suggests that human keratin-associated protein 1 family genes may have arisen mainly through gene duplication of the cysteine-repeat motifs during evolution.

Characterization of a First Domain of Human High Glycine-Tyrosine and High Sulfur Keratin-associated Protein (KAP) Genes on Chromosome 21q22.1

Analysis of the EBI/GeneBank(TM) data base using non-human hair keratin-associated protein (KAP) cDNA sequences as a query resulted in the identification of a first domain of high glycine-tyrosine and high sulfur KAP genes located on human chromosome 21q22.1. This domain, present on the DNA accession numbers and, was approximately 535 kb in size and contained 17 high glycine-tyrosine and 7 high sulfur KAP genes, as well as 9 KAP pseudogenes. Based on amino acid sequence comparisons of the encoded proteins, the KAP genes could be divided into seven high glycine-tyrosine gene families (KAP6-KAP8, and KAP19-KAP22) and four high sulfur gene families (KAP11, KAP13, KAP15, and KAP23). The high glycine-tyrosine genes described here appear to represent the complete set of this type of KAP genes present in the human genome. Both systematic cDNA isolation studies from an arrayed scalp cDNA library and in situ hybridization expression studies of all of the KAP genes identified in the 21q22.1 region revealed varying degrees and regions of expression of 11 members of the high tyrosine-glycine genes and 6 members of the high sulfur KAP genes in the hair forming compartment.

Characterization of a Cluster of Human High/Ultrahigh Sulfur Keratin-associated Protein Genes Embedded in the Type I Keratin Gene Domain on Chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.

Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia.

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.

Pmg-1 and Pmg-2 constitute a novel family of KAP genes differentially expressed during skin and mammary gland development

The epidermis, by invagination of the undifferentiated ectodermal cells, gives rise to several distinct structures including hair, sebaceous, eccrine sweat and mammary glands. We have recently isolated a novel gene, pmg-1, expressed in the pubertal mouse mammary gland. While investigating its genomic structure, we identified a related gene in close proximity, which we have termed pmg-2. pmg-1 and pmg-2 are intron-less, are transcribed in opposite directions and are separated by a potential promoter region of 2.8 kb containing putative binding motifs for the developmental transcription factors Lef-1, Sox5 and D-STAT. pmg-1 and pmg-2 encode small proteins rich in G, S, F, Y and Q and contain characteristic repeats reminiscent of the keratin-associated proteins (KAPs). Both genes are expressed in growing hair follicles in skin as well as in sebaceous and eccrine sweat glands. Interestingly, expression is also detected in the mammary epithelium where it is limited to the onset of the pubertal growth phase and is independent of ovarian hormones. Their broad, developmentally controlled expression pattern, together with their unique amino acid composition, demonstrate that pmg-1 and pmg-2 constitute a novel KAP gene family participating in the differentiation of all epithelial cells forming the epidermal appendages.

A Cluster of Keratin-Associated Proteins on Mouse Chromosome 10 in the Region of Conserved Linkage with Human Chromosome 21

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes,Krtap12-1,has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein.Krtap12-1is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family.Krtap12-1is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, betweenITGB2(integrin β2) andPFKL(the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.

Isolation and Characterization of a Putative Keratin-Associated Protein Gene Expressed in Embryonic Skin of Mice

Embryonic mouse skin undergoes substantial morphologic changes from 13 days post-coitus (dpc) to 16 dpc, i.e., from simple layers of epithelial cells and periderm at 13.5 dpc to almost fully differentiated stratified epithelium with the rudiments of hair follicles at 16.5 dpc. Using RNA differential display, we isolated a gene involved in the development of mouse epidermis. This gene, tentatively designated as 4C32, encodes 197 amino acids containing six direct repeats of 10 amino acids with the CQ motif. The repetitive structure with the CQ motif is seen in most keratin-associated protein families, which are known to be specifically expressed in hair follicles. 4C32 is expressed in the outermost layer of the embryonic epidermis at 15.5 and 16.5 dpc, and abruptly disappears at 17.5 dpc, suggesting that 4C32 is expressed in the periderm. The periderm is a superficial layer of embryonic epidermis, and is known to disappear at 17 dpc in mouse embryos. The 4C32 transcripts were also detected in the developing and matured tongue tissues and in the tail scale, but not at any stage in hair follicles.

Expression of a wool intermediate filament keratin transgene in sheep fibre alters structure.

Alteration of the protein composition of the wool fibre via transgenesis with sheep wool keratin and keratin associated protein (KAP) genes may lead to production of fibre types with improved processing and wearing qualities. Using this approach, we have demonstrated that high level cortical-specific expression of a wool type II intermediate filament (IF) keratin gene, K2.10, leads to marked alterations in both the microstructure and macrostructure of the wool fibres, which have higher lustre and reduced crimp. Analysis of mRNA found reduced levels of transcripts from endogenous cortical type I (p < 0.05) and type II (p < 0.01) keratin IF genes and from the KAP8 (p < 0.001) and KAP2 (p < 0.01) gene families. Examination of protein composition revealed an altered ratio in the keratin type II protein family of the wool fibre cortex. Whilst the over-expressed K2.10 transgene product constituted the majority of keratin type II IF protein, it appeared unable to form heterodimers with much of the expressed endogenous keratin type I IF. In comparison with non-transgenic sheep, fewer IF microfibrils were visible in the cortical cells of fibres from transgenics. The combined effect on fibre structure was disruption of the formation of orthocortical and paracortical cells in the fibre cortex, a factor which could account for the reduction in fibre crimp. No effects upon transcript or protein levels, or fibre microstructure or macrostructure were observed in transgenic sheep expressing the transgene at lower levels, indicating that subtle changes to the gene expression profile in sheep wool follicles can be tolerated. The data here also illustrate that control over endogenous transcript levels in the cortex results when factors acting on the endogenous keratin type I, keratin type II and KAP gene sequences are sequestered by the active K2.10 transgene locus. Moreover, interference to a transcriptional hierarchy shared by keratin and KAP genes may occur prior to establishment of the orthocortical and paracortical compartments of the follicle cortex, at the level of the chromatin.

Linkage mapping of wool keratin and keratin-associated protein genes in sheep.

Linkage mapping of wool keratin and keratin-associated protein genes in sheep.

Linkage Relationships between Keratin-Associated Protein (KRTAP) Genes and Growth Hormone in Sheep

The ovine hair keratin-associated protein (KRTAP) genes are grouped into several families. In this study, we analyzed linkage relationships between members of four KRTAP families and growth hormone and β-hemoglobin. Restriction fragment length polymorphisms and a microsatellite polymorphism were typed in 10 sire groups from a flock of Australian Merino sheep. Two linkages, both previously undetected in sheep or any other mammal, were found. They were between growth hormone (GH) and the high-sulfur keratin gene family KRTAP1 (previously called high-sulfur B2C) at θ = 0.10, Z = 3.01 and between two high-glycine-tyrosine class genes, KRTAP6 (previously high-glycine-tyrosine type II) and KRTAP8 (previously high-glycine-tyrosine type I-F), at θ - 0.15, Z = 5.0. The high level of conservation of the mammalian genome at these recombination values suggests that these linkages might be found in distantly related mammalian species.

Evidence of linkage between high‐glycine‐tyrosine keratin gene loci and wool fibre diameter in a Merino half‐sib family

Candidate genes for quantitative trait loci have been studied in a Medium Peppin Merino flock. Obvious candidates for effects on wool production traits are genes for the major proteins expressed in the wool fibre, the keratin and keratin-associated protein genes. Two keratin-associated protein loci, KRTAP6 and KRTAP8, have previously been shown to be linked. The results of analyses between these two loci and production traits gave significant evidence of linkage with wool fibre diameter in one out of eight half-sib groups tested. High-glycine-tyrosine proteins (KRTAP6, 7 and 8) are known to vary considerably in abundance in wool fibres and it is possible that a gene for major effect on fibre diameter is located within the same chromosomal region as KRTAP6 and KRTAP8.

Organization and expression of hair follicle genes

Several families of proteins are expressed in the growth of hair and an estimated 50–100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin.A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/ tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para-and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hairloss mice.Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para-to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought.

Organization and expression of hair follicle genes.

Several families of proteins are expressed in the growth of hair and an estimated 50-100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin. A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para- and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hair-loss mice. Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para- to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought.